Studies On The Material Basis For Efficacy Of Oyster Shells Coupled With Radix Puerariae

Oyster shells are the shells of Ostrea gigas Thunberg,Ostrea talienwhanensis Crosse,or Ostrea rivuLaris GouLd.It is a commonly used medicine in traditional Chinese Medicine,and is contained in Chinese Pharmacopoeia.It is salty and slight cold in nature,belonging to liver,gallbladder,and kidney channels.The raw oyster shells have the functions of tranquilization with heavy material,exuberanting yang and replenishing yin,and dispersing clumping.The oyster shells contain over 90%calcium carbonate,and the organic matrix is composed of different macromolecules including proteins and polysaccharides.Currently the primary pharmacological studies on oyster shells include sedation,inhibition of spontaneous activity,and hypnotic effect.At present the in vivo studies are primarily performed,but the organic matrix components and their pharmacological actions have not been yet reported.In the clinic,oyster shells are often used in couple with Radix Puerariae for the treatment of hypotension.Given that the major bioactive components in Radix Puerariae have been identified and the underlying mechanisms have been explored,the current study therefore focus on the material basis for the pharmacological effects of oyster shells coupled with Radix Puerariae.The results obtained in this study would be of significance for the development of shellfish marine drugs and modernization of traditional Chinese medicines.The present study analyzed the basic chemical components of oyster shells.We established the fingerprinting of the inorganic components using x ray diffraction,and analyzed the organic components via ion-exchange column chromatography and reversed C8 column chromatography for separation,and resultantly oligopeptides components were obtained.We examined the sedative effect of pharmacological material basis,and then studied the pharmacological material basis for oyster shells coupled with Radix Puerariae,including the changes after compatibility in physical and chemical properties,the effects of Radix Puerariae on Ca2+ dissolution of oyster shells,and the effects of oyster shells on the bioactive material dissolution of Radix Puerariae.We also investigated the pharmacological actions of oyster shells coupled with Radix Puerariae and the underlying mechanisms,including sedative and anti-hypertensive effects.Finally,we explored the pharmacokinetics of puerarin using HPLC-MS in rats,and also investigated the interactions between puerarin and drug-metabolizing enzymes and transport proteins in vitro using spectroscopy and molecular docking strategies.The first part is literature research.We reviewed the recent studies on oyster shells and Radix Puerariae,and also we summarized advances in studies on effective substance basis of Chinese compound formula.This part provides approaches and methods for the current studies.The second part studied the chemical components of oyster shells.We analyzed the fingerprinting of inorganic components using differential thermal analysis,infrared spectrum and x ray diffraction.Calcium carbonate was identified as the major component and it was degraded to calcium oxide with the increase in temperature.The XRD fingerprinting of oyster shells had good specify.The allophanamide assay was used to quality and quantify the organic components in oyster shells and oligopeptides were identified to account for 38.02%.The phenol-sulfuric acid method was used to analyze the content of total sugars.Amino acid composition analysis was further used to analyze the organic components.Ion exchange chromatography was used to preliminarily to separate and purify the organic components and the molecular weight range was determined to be less than 1000 using mass spectrum.The reversed C8 column chromatography was further performed to obtain the oligopeptides and the purity was determined by HPLC-MS.We finally investigated the anti-oxidant effects of the oligopeptides,showing that they had good anti-oxidant properties and eliminated the free radicals and superoxide anion dose-dependently.The third part studied the sedative effects of oyster shells.We examined the effects of oyster shells,calcium carbonate,and the organic components on the sleeping time induced by pentobarbital sodium in mice,and explored the material basis for the sedative effects.The data showed that single dose of oyster shells had no sedative effect,but continuous treatment led to significant sedative effect in a dose-dependent manner,suggesting that the sedative effect of oyster shells had superposition.Moreover,calcium carbonate also showed significant sedative effects like raw oyster shells.However,the organic components of raw oyster shells showed relative week capacity in synergy with pentobarbital sodium for inducing the sleeping time.These results indicated that the sedative effect of oyster shells was associated with calcium carbonate.Furthermore,western blot assays demonstrated that the expression of 5-HT receptor was downregulated and the expression of GABA receptor was upregulated in mice brain,suggesting that the sedative effects of oyster shells were possibly attributed to the modulation of the neurotransmitter receptors and their transduction in brain.The fourth part studied the pharmacological material basis for efficacy of oyster shells coupled with Radix Puerariae.We first examined the conductivity,salinity,viscosity,turbidity,and pH of water extract,showing that the increase in the proportion of oyster shells reduced the conductivity,salinity,viscosity,TDS and turbidity,but increased the pH.The changes in viscosity and turbidity indicated that the compatibility of the two drugs could enhance the solubility of the non-soluble components.The changes in conductivity and salinity suggested that the flavonoids compounds in Radix Puerariae could exert their effects via forming complex with the calcium ion dissoluted from oyster shells.Then,we studied the effects of Radix Puerariae on calcium dissolution from oyster shells,showing that increased proportion of Radix Puerariae increased the Ca content,suggesting that Radix Puerariae promoted the Ca dissolution from oyster shells.Meanwhile,the effect was reversed if oyster shells were replaced with calcium carbonate,indicating that the chemical calcium carbonate could not be used to replace oyster shells.Finally,we studied the effects of oyster shells on the dissolution of pharmacological materials from Radix Puerariae,showing that oyster shells significantly promoted the dissolution of pharmacological materials from Radix Puerariae.Similarly,if oyster shells were replaced with calcium carbonate,the dissolution of puerarin and total puerariae flavones was weakened,also suggesting that calcium carbonate could not be used to replace oyster shells.The fifth part studied the pharmacological actions of oyster shells coupled with Radix Puerariae.We first examined the sedative effect by determining the sleeping time induced by pentobarbital sodium in mice,showing that the combination use of two drugs had more significant sedative effects than the single dese.Western blot assays furthered demonstrated that oyster shells combined with Radix Puerariae could significantly down-regulated the expression of 5-HT receptor and up-regulated the expression of GABA receptor in mice brain,suggesting the sedative effects were associated with regulation of neurotransmitter receptors.On the other hand,we investigated the anti-hypertensive effects of using spontaneous hypertensive rats.We found that oyster shells,Radix Puerariae and their combination could influence the blood pressure,serum calcium,vascular smooth muscle intracellular free calcium,NO,endothelin,atrial natriuretic factor,renin,angiotensin ?,aldosterone,hemorheology,and left ventricle.These effects could be associated with the anti-hypertensive effects of oyster shells coupled with Radix PuerariaeThe sixth part studied the pharmacokinetics of puerarin using HPLC-MS in rats,and established the HPLC-MS methods.We used electrospray ion source and multiple reaction monitoring mode with rutin as the internal standard.The DAS 2.0 software was used to process the pharmacokinetics data and the time-concentration curve of puerarin was obtained.The curve showed that the area under time-concentration curve was larger and the peek had high intensity,indicating that by the synergistic effects of oyster shells,puerarin was largely and rapidly absorbed into blood circulation in rats.Oral administration of the water extract of oyster shells coupled with Radix Puerariae led to large apparent volume of distribution,suggesting that puerarin was largely distributed into the tissues and had good affinity to tissues or proteins under the synergism of oyster shells.Besides,oral administration of the water extract showed long half-time and mean residence time,indicating that puerarin was eliminated slowly in the presence of oyster shells.All these data suggested that oyster shells had considerable effects on the metabolism of the pharmacological materials contained in Radix Puerariae,and could promote the absorption and distribution of puerarin.The seventh part investigated the interactions between puerarin and drug-metabolizing enzymes and transport proteins in vitro as well as the effects of calcium ion.We used fluorescence spectrum,ultraviolet spectrum,and analysis of round two chromatography to examine the interaction between puerarin and cytochromes CYP1A2 and CYP2D6.Meanwhile,the Autodock software was used to perform the molecular docking studies.The obtained results demonstrated that puerarin quenched CYP1A2 and CYP2D6 via a mechanism of static quenching.Hydrophobic interaction and hydrogen bond were the major binding force.Puerarin could change the conformation of CYPs and calcium ion could enhance the affinity of Puerarin to CYPs.We also used fluorescence spectrum,ultraviolet spectrum and nuclear magnetic resonance to investigate the interaction between puerarin and human serum albumin.The results showed that a mechanism of static quenching also existed and the major binding force was also hydrophobic interaction and hydrogen bond.Puerarin could change the conformation of HSA and calcium ion could enhance the affinity of Puerarin to HSA.These data were consistent with the pharmacokinetic results presented in last chapter.In summary,calcium carbonate is the primary material basis for the efficacy of oyster shells.The chemical calcium carbonate could not be used to completely replace oyster shells for pharmacological use.Oyster shells coupled with Radix Puerariae could promote the dissolution of pharmacological materials from drugs.The combination use of the two drugs resulted in more significant sedative and anti-hypertensive effects.The current data provide novel insights into the chemistry and pharmacology of shellfish medicines and their combination with medicinal herbs for enhancing efficacy.